Amino acid hydrolysate standard

V. alginolyticus exopolysaccharide (1% w/v) in water was hydrolyzed by addition of an equal volume of 11 N HCl for 5 h at 105 °C. After a cooling step, neutralization was carried out with 10 N NaOH, and then with 1 N NaOH, using pH-indicator paper to reach pH 6–7. The final volume was adjusted to 25 mL with ultrapure water and filtered on a 0.2 µm sterilizing grade membrane.
The neutralized samples and an amino acid hydrolysate standard (Waters) were derivatized by adding 6-aminoquinolyl-N-hydroxysuccinimidyl carbamate using the AccQ-Ta Ultra Derivatization kit (Waters). The derivatized samples (10 µL) were injected on a AccQ-Tag Ultra C18 column (1.7 µm 2.1 × 100 mm, Waters) that was mounted on a Waters Acquity H class ultra-high-performance liquid chromatography apparatus that was equipped with UV detector working at 260 nm.
A gradient elution was conducted for 10 min at 55 °C starting with 100% eluent A (acetonitrile/formic acid) to 100% of eluent B (acetonitrile). Signals were compared with derivatized amino acid standards. According to experimental conditions, serine and alanine eluted at 2.3 min and 4.4 min, respectively.

The whole body adult whiteflies were homogenized for amino acid analysis by UPLC using the protocol described previously [25 (link), 56 (link)]. Briefly, samples were injected into an Agilent UPLC with a PDA detector and AccQ-Tag Ultra 2.1 x 100 mm column. Amino acids are determined by comparing their retention time with standards, protein-amino acids μl^-1 (Waters amino acid hydrolysate standard #088122, supplemented with asparagine, tryptophan, and glutamine) and quantified with standard curves. Proteins were quantified using a Lowry Protein Assay Kit (Sangon, Biotech) following manufacturer’s instructions using bovine serum albumin as a standard. Amounts of individual amino acids were normalized to the total protein content.